Project Summary Anaplastic thyroid cancer (ATC) is one of the deadliest human malignancies with median survival of < 6 months despite treatment with a combination of surgery, external beam radiation and cytotoxic chemotherapy. Paclitaxel and docetaxel are cytotoxic drugs that work by stabilizing mitotic spindles and increasing the rate of chromosomal segregation errors during cell division. Taxanes have shown a modest benefit in patients with ATC, but even with treatment, patient survival remains very poor. Recently, the combination of dabrafenib and trametinib have shown efficacy in patients with BRAFV600E mutated tumors, and this combination has been approved by the FDA for use in patients with this specific mutation. We recently published mutation status of the largest group of patients with ATC (Pozdeyev N, Clin Cancer Res, 2018). Of 196 patients, 41% had a BRAF mutation, leaving 59% of patients not eligible for the dabrafenib/trametinib therapy. More effective drugs and rational synergistic drug combinations are urgently needed for these patients. We have used a novel functional genomics discovery approach to identify the spindle assembly checkpoint (SAC) pathway as a key synergistic lethal vulnerability with taxanes in ATC. We have also shown that ATC and poorly differentiated thyroid cancer (PDTC) tumors have high levels of critical SAC components, MPS1 and BUBR1. The primary objective of this application is to study the combination of taxanes with the drugs blocking the SAC pathway for the treatment of ATC. Our central hypothesis is that the inhibition of the SAC pathway, responsible for the prevention of chromosomal segregation errors in ATC and PDTC cells, which already have high baseline chromosomal instability (CIN), will sensitize a vulnerable subset of tumor cells to taxanes and cause massive aneuploidy incompatible with cell survival. We further predict that the aneuploidy generated by this therapy will activate the immunosurveillance system, which we will test in an immunocompetent preclinical model. We will test these hypotheses using in vitro human ATC/PDTC cells lines to elucidate mechanisms of synergy sensitivity and resistance, validate these findings in orthoptopic xenograft models, and extend these studies into syngeneic orthotopic models to determine the contribution of immunosurveillance on effectiveness of therapy. Successful completion of these Aims will determine the preclinical efficacy of combination taxane therapy and SAC pathway inhibition in ATC and PDTC. These studies will also provide mechanistic insights that could lead to other novel combination therapies and critical biomarkers for focused clinical trials. If successful, this work should rapidly lead to clinical trials that will improve survival in these patients who desperately need better therapies. These studies will likely also inform treatments in other advanced cancers. `